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Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori
Author(s) -
Uno Tomohide,
Nakada Takuya,
Okamaoto Sota,
Nakamura Masahiko,
Matsubara Mamoru,
Imaishi Hiromasa,
Yamagata Hiroshi,
Kanamaru Kengo,
Takagi Michihiro
Publication year - 2007
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.20201
Subject(s) - biology , phosphorylation , rab , kinase , biochemistry , bombyx mori , protein kinase a , antiserum , fusion protein , protein phosphorylation , microbiology and biotechnology , gtpase , antibody , gene , recombinant dna , immunology
The Rab family of small GTPases are key regulators of membrane trafficking. Partially purified Rab8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C in mammalian cells in vitro. To determine which of the seven serines and four threonines are phosphorylated, we generated deletion and site‐directed mutants of BRab8, inserted them in Escherichia coli , partially purified the encoded fusion proteins by affinity chromatography, and examined their phosphorylation by protein kinase C in vitro. We found that Ser‐132 of BRab8 was specifically phosphorylated by protein kinase C. In addition, Western blotting using an antiserum against BRab8 and in‐gel staining for phosphorylated proteins revealed that BRab8 is phosphorylated in vivo. Arch. Insect Biochem. Physiol. 66:89–97, 2007. © 2007 Wiley‐Liss, Inc.